Memory B Cells Encode Neutralizing Antibody Specific for Toxin B from the Clostridium difficile Strains VPI 10463 and NAP1/BI/027 but with Superior Neutralization of VPI 10463 Toxin B

Abstract

T he role of immune cell memory in Clostridium difficile infection (CDI) remains poorly understood. CDI is complicated by a high frequency of recurrence, often after disease has apparently resolved, and can be associated with progressively worsening pathology and ultimately death (1). However, patients that develop antibodies (Ab) capable of neutralizing two toxins secreted by C. difficile (TcdA and TcdB) are less likely to experience recurrence (reviewed in reference 2). This suggests that memory B (Bmem) cells may contribute to resistance to reinfection by encoding toxin-neutralizing Ab. Bmem cells have typically undergone affinity maturation and Ab class switch in the germinal centers of secondary lymphoid organs (3). Bmem cells are therefore poised to respond rapidly to booster vaccinations or infection, by rapidly differentiating into plasma cells that secrete class-switched, highaffinity Ab (4). Such plasma cells may display a range of short-toextreme longevity, be associated with transient or sustained Ab titers, or secrete neutralizing or nonneutralizing Ab (5-7).In earlier studies, a correlation between bacterial load and advanced age was observed during CDI, with older individuals lacking toxin-neutralizing Ab (8). In more recent work, the probability of HIV-positive patients experiencing CDI increased as their CD4 ϩ T-cell counts declined (9), which could be partly attributable to altered CD4 ϩ T-cell-dependent B cell function (10). Indeed, there is growing concern about CDI in a variety of immunocompromised individuals, including organ transplant recipients (reviewed in reference 11). These observations highlight the wellrecognized importance of B cell responses and production of toxin-neutralizing antibodies in resisting CDI. However, the underlying characteristics of the Bmem cellular response and their contributions to production of toxin-neutralizing Ab have not been described.CDI is best known as a disease of the gastrointestinal tract, causing diarrhea, and this infection may progress to a severe pathological condition in which pseudomembranous colitis and toxic megacolon are evident (1, 2). However, CDI-associated mortality may be attributable to systemic sequelae of the disease (12). Although large-scale epidemiological studies are lacking, reported systemic complications include hepatic abscesses (13), ascites (14), pleural effusion with acute respiratory distress (15, 16), and severe sepsis and multiorgan dysfunction (17).TcdA and TcdB are large clostridial toxins that contribute sub-

Deletion of a 19-Amino-Acid Region in Clostridioides difficile TcdB2 Results in Spontaneous Autoprocessing and Reduced Cell Binding and Provides a Nontoxic Immunogen for Vaccination

“…Mice were immunized at between the ages of 8 and 10 weeks with a subcutaneous injection divided between both rear flanks. Vaccines consisted of 50 g of TcdB2 D270N or TcdB2 Δ1769 -1787 in sterile PBS adsorbed to alhydrogel alum (InvivoGen, San Diego, CA) (35). The mice were initially vaccinated on day 0, followed by a booster at day 28.…”

Adaptive immune constraints on C. difficile vaccination

“…There could also be intrinsically different adaptive immune responses to similar Ags produced by different strains. The Lang laboratory recently reported that TcdB1 and TcdB2 were similarly immunogenic in mice, resulting in very similar Ab profiles with regard to Ig sub-class [10]. In that study, while long-term memory B-cell driven Ab recall responses were observed against TcdB1 and TcdB2, neutralization of TcdB2 and resistance to in vivo challenge with toxin was vastly inferior to that against TcdB1 [10].…”

“…The Lang laboratory recently reported that TcdB1 and TcdB2 were similarly immunogenic in mice, resulting in very similar Ab profiles with regard to Ig sub-class [10]. In that study, while long-term memory B-cell driven Ab recall responses were observed against TcdB1 and TcdB2, neutralization of TcdB2 and resistance to in vivo challenge with toxin was vastly inferior to that against TcdB1 [10]. This suggests that the memory B cell compartment could essentially be mis-directed against TcdB from some strains, resulting in high titers of non-neutralizing Ab.…”

“…Alum is heavily used in the US and results in reasonably good humoral immunity, but stimulates poor cellular immunity [15]. However, Alum formulations substantially enhance the anti-TcdB IgG response in mouse models [10], but the benefit of adjuvant inclusion is less clear in human volunteers [16]. Another major challenge is the inherent variation in human immune responses.…”

“…It is known that individuals with a prior C. difficile infection have circulating memory B cells reactive to TcdA and TcdB [10,19]. A single cell analysis of the Ab repertoire may offer important insights into responses that correlate with toxin neutralization and/or limited disease recurrence.…”